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Steatohepatitis and Vascular Thrombosis in Apolipoprotein E Deficient Mice
Thrombosis Research 129 (2012) e166–e167
Contents lists available at SciVerse ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Letter to the Editors-in-Chief Steatohepatitis and Vascular Thrombosis in Apolipoprotein E Deficient Mice Dear Editors, Obesity is a risk factor for common vascular complications including myocardial infarction[1], stroke [2] and venous thrombosis[3]. Since the acute, proximal cause of these vascular complications is thrombosis, obesity may promote the formation of vascular thrombosis. However, not all states of obesity are associated with increased risk of vascular complications, rather it is the visceral obesity associated with features of the metabolic syndrome that is particularly associated with the risk for vascular events[4]. In addition, inflammatory markers are associated with components of the metabolic syndrome and vascular risk [5]. The mechanism(s) by which obesity induces a systemic inflammatory state may be multifactorial with important contributions from the liver since fatty infiltration of the liver is very common in obesity. For example, the presence of fatty liver disease was 93% in a series of patients undergoing bariatric surgery with 26% of these patients also exhibiting signs of steatohepatitis [6]. Since many of the inflammatory factors elevated in obesity, such as C-reactive protein, are produced by the liver [7], it may be that the inflammatory liver disease associated with obesity [8] is playing a role in the vascular complications associated with obesity. To determine the potential for liver injury to promote vascular disease in mice, we treated apolipoprotein E deficient mice (ApoE−/−) with carbon tetrachloride (CCL4), a well-established hepatotoxin that causes hepatic steatosis, inflammation, and fibrosis of liver tissue [9,10]. A 1:100 dilution of CCL4 in corn oil was given by intraperitoneal (IP) injection to experimental mice weekly for six weeks (n= 10) while control mice (n = 10) were treated with IP injections of the corn oil vehicle. Mice were fed a western diet (TD88137, Harland Teklad, Madison, MI) throughout the protocol in attempts to accelerate atherosclerosis so that the effects of liver injury could be determined towards both atherosclerosis and thrombosis. In addition, both high fat diet and deficiency of apolipoprotein E have been shown to exacerbate liver injury induced by CCL4, possibly due to increased generation of oxysterols [9]. 24 hours following the last injection of drug or vehicle, alanine aminotransferase levels (9326.7 ± 2341.5 vs 39.0 ± 7.0 U/I, p b 0.002), lactate dehydrogenase levels (35110.7 ± 7538.0 vs 595.0 ± 57.8 U/I, p b 0.001) and monocyte chemoattractant protein-1 levels (504.5 ± 77.6 vs 165.0 ± 38.0 pg/ml, p b 0.005) were all elevated in CCL4 treated mice compared to vehicle treated mice, respectively. Histological analysis of the liver revealed greater macrophage content (4.0± 0.4 vs 0.7 ± 0.2% Mac3 positive staining area, p b 0.001, Fig. 1A and B), and increased collagen content (2.3 ± 0.4 vs 1.1 ± 0.3% positive staining area using Sirius red, p b 0.03) in mice treated with CCL4 compared to control mice. There were no differences in body weight throughout the protocol indicating CCL4 did not cause illness to the point of weight loss. Despite clear evidence of liver injury, there was no effect of chronic (6 weeks) CCL4 treatment on atherosclerosis surface area when measured by Oil-red-O (2.1 ± 0.3 vs 1.8 ± 0.3% surface coverage of aortic 0049-3848/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.thromres.2012.01.010
tree and major branches, p = NS) or by lesion cross sectional analysis at the aortic valve (1064.1 ± 159.5 vs 1142.2 ± 144.4 μm2, p = NS). To determine whether the liver injury induced by CCL4 would affect macrovascular arterial thrombosis at a distant site, photochemical injury was performed to the mid common carotid artery using Rose Bengal [11]. This model produces endothelial injury followed by occlusive thrombosis and the time to occlusion is used as a measure of thrombotic tendency [11]. Mice receiving CCL4 treatment formed occlusive thrombosis in a shorter period of time compared to vehicle control mice (Fig. 1C) indicating that chronic CCL4-induced liver injury leads to a prothrombotic state. This prothrombotic effect was independent of atherosclerosis as no differences in atherosclerosis were detected between mice receiving CCL4 or vehicle control. The relationship between liver disease and thrombosis is complicated as the liver is a source of inflammatory, coagulant, and anticoagulant factors [12]. Several types of liver diseases have been associated with both hemostatic defects and thrombosis [12]. In the case of steatohepatitis associated with obesity, the balance between bleeding and thrombosis is more in favor of thrombosis[13,14] and this may explain the increased risk of thrombotic events in patients with the metabolic syndrome. This study demonstrates that liver injury is sufficient to enhance thrombosis following arterial injury. Multiple factors may contribute to this risk including alterations in circulating procoagulant and antifibrinolytic factors. Interestingly, plasma levels of fibrinogen (Fg) (5.0 ± 2.0 vs 5.4 ± 1.1 mg/ml, p = NS) and plasminogen activator inhibitor-1 (PAI-1) (20.7 ± 10.3 vs 14.5 ± 3.4 ng/ml, p = NS) were not different between control mice and mice receiving CCL4, respectively. However, thrombinantithrombin complexes (TATs) were elevated in CCL4 treated mice compared to control mice (Fig. 1D) indicating that thrombin generation was increased (or anti-coagulant factors were decreased) in this model of steatohepatitis. Consistent with the hypothesis that thrombin generation is involved in steatosis, it was recently reported in a model of dietinduced obesity that TAT's are elevated with onset of steatosis [15]. Although this model of liver injury is not induced by obesity, it is an important step in determining the potential independent role of liver abnormalities on thrombosis, as models of obesity are confounded by many extrahepatic factors that could also contribute to thrombotic risk. However, even with this model we cannot rule out the possibility that extrahepatic effects of CCL4 are contributing to thrombosis. In this regard, it will be useful to determine the effects of other forms of hepatic injury on arterial thrombosis. In summary, hepatic steatosis induced by chronic CCL4 leads to enhanced arterial thrombosis. Changes in liver function coincident with disease states such as visceral obesity may contribute to the increased vascular risk associated with these diseases. Therapeutic strategies targeting coagulation-related pathways may be beneficial in reducing the prothrombotic state associated with steatohepatitis. Disclosure of conflict of interests The authors have no conflict of interests.
Letter to the Editors-in-Chief
A
Control injection
e167
B
CCl4 injection
Mac-3 staining (400x)
D *
80
*
3
TAT (ng/ml)
Time to Occlusion (min)
C
60 40 20 0
2 1 0
Control
CCl4
Control
CCl4
Fig. 1. Macropahge staining of liver tissue with Mac-3 anti-mouse antibody following (A) control oil or (B) CCL4 injection, magnification = 400x. Scale bar = 100 μm. (C) Bars represent time to occlusive carotid thrombosis in control mice (white bar) compare to mice receiving CCL4 (black bar) (mean ± SEM). *p b 0.003. (D) Bars represent thrombinantithrombin complexes in plasma from control (white bar) and CCL4-treated (black bar) mice (mean ± SEM). *p b 0.0001.
Acknowledgements This work was supported by the National Institutes of Health (HL073150 to D.T.E.) and the Veterans Administration Merit Review Award (1I01BX000353 to D.T.E.). References [1] Yusuf S, Hawken S, Ounpuu S, Bautista L, Franzosi MG, Commerford P, et al. Obesity and the risk of myocardial infarction in 27,000 participants from 52 countries: a case–control study. Lancet 2005;366:1640–9. [2] Yatsuya H, Folsom AR, Yamagishi K, North KE, Brancati FL, Stevens J. Race- and sex-specific associations of obesity measures with ischemic stroke incidence in the Atherosclerosis Risk in Communities (ARIC) study. Stroke 2010;41:417–25. [3] Borch KH, Braekkan SK, Mathiesen EB, Njolstad I, Wilsgaard T, Stormer J, et al. Anthropometric measures of obesity and risk of venous thromboembolism: the Tromso study. Arterioscler Thromb Vasc Biol 2010;30:121–7. [4] Zirlik A, Abdullah SM, Gerdes N, MacFarlane L, Schonbeck U, Khera A, et al. Interleukin-18, the metabolic syndrome, and subclinical atherosclerosis: results from the Dallas Heart Study. Arterioscler Thromb Vasc Biol 2007;27:2043–9. [5] Ridker PM, Buring JE, Cook NR, Rifai N. C-reactive protein, the metabolic syndrome, and risk of incident cardiovascular events: an 8-year follow-up of 14 719 initially healthy American women. Circulation 2003;107:391–7. [6] Ong JP, Elariny H, Collantes R, Younoszai A, Chandhoke V, Reines HD, et al. Predictors of nonalcoholic steatohepatitis and advanced fibrosis in morbidly obese patients. Obes Surg 2005;15:310–5. [7] Sun H, Koike T, Ichikawa T, Hatakeyama K, Shiomi M, Zhang B, et al. C-reactive protein in atherosclerotic lesions: its origin and pathophysiological significance. Am J Pathol 2005;167:1139–48. [8] Targher G, Bertolini L, Padovani R, Zenari L, Zoppini G, Falezza G. Relation of nonalcoholic hepatic steatosis to early carotid atherosclerosis in healthy men: role of visceral fat accumulation. Diabetes Care 2004;27:2498–500. [9] Ferre N, Martinez-Clemente M, Lopez-Parra M, Gonzalez-Periz A, Horrillo R, Planaguma A, et al. Increased susceptibility to exacerbated liver injury in hypercholesterolemic ApoE-deficient mice: potential involvement of oxysterols. Am J Physiol Gastrointest Liver Physiol 2009;296:G553–62.
[10] Luster MI, Simeonova PP, Gallucci RM, Matheson JM, Yucesoy B. Immunotoxicology: role of inflammation in chemical-induced hepatotoxicity. Int J Immunopharmacol 2000;22:1143–7. [11] Westrick RJ, Winn ME, Eitzman DT. Murine models of vascular thrombosis (Eitzman series). Arterioscler Thromb Vasc Biol 2007;27:2079–93. [12] Lisman T, Caldwell SH, Burroughs AK, Northup PG, Senzolo M, Stravitz RT, et al. Hemostasis and thrombosis in patients with liver disease: the ups and downs. J Hepatol 2010;53:362–71. [13] Cigolini M, Targher G, Agostino G, Tonoli M, Muggeo M, De Sandre G. Liver steatosis and its relation to plasma haemostatic factors in apparently healthy men–role of the metabolic syndrome. Thromb Haemost 1996;76:69–73. [14] Villanova N, Moscatiello S, Ramilli S, Bugianesi E, Magalotti D, Vanni E, et al. Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease. Hepatology 2005;42:473–80. [15] Kassel KM, Owens III AP, Rockwell CE, Sullivan BP, Wang R, Tawfik O, et al. Proteaseactivated receptor 1 and hematopoietic cell tissue factor are required for hepatic steatosis in mice fed a Western diet. Am J Pathol 2011;179:2278–89.
W. Luo M. Öhman A. Wright S. Kamrudin H. Wang C. Guo D. Eitzman⁎ University of Michigan, Department of Internal Medicine, Cardiovascular Research Center, Ann Arbor, Michigan, USA ⁎Corresponding author at: University of Michigan, Cardiovascular Research Center, 7301A MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109–0644, USA. Tel.: + 1 734 647 9865; fax: +1 734 936 2641. E-mail address: [email protected].(D. Eitzman) 28 November 2011
Contents lists available at SciVerse ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Letter to the Editors-in-Chief Steatohepatitis and Vascular Thrombosis in Apolipoprotein E Deficient Mice Dear Editors, Obesity is a risk factor for common vascular complications including myocardial infarction[1], stroke [2] and venous thrombosis[3]. Since the acute, proximal cause of these vascular complications is thrombosis, obesity may promote the formation of vascular thrombosis. However, not all states of obesity are associated with increased risk of vascular complications, rather it is the visceral obesity associated with features of the metabolic syndrome that is particularly associated with the risk for vascular events[4]. In addition, inflammatory markers are associated with components of the metabolic syndrome and vascular risk [5]. The mechanism(s) by which obesity induces a systemic inflammatory state may be multifactorial with important contributions from the liver since fatty infiltration of the liver is very common in obesity. For example, the presence of fatty liver disease was 93% in a series of patients undergoing bariatric surgery with 26% of these patients also exhibiting signs of steatohepatitis [6]. Since many of the inflammatory factors elevated in obesity, such as C-reactive protein, are produced by the liver [7], it may be that the inflammatory liver disease associated with obesity [8] is playing a role in the vascular complications associated with obesity. To determine the potential for liver injury to promote vascular disease in mice, we treated apolipoprotein E deficient mice (ApoE−/−) with carbon tetrachloride (CCL4), a well-established hepatotoxin that causes hepatic steatosis, inflammation, and fibrosis of liver tissue [9,10]. A 1:100 dilution of CCL4 in corn oil was given by intraperitoneal (IP) injection to experimental mice weekly for six weeks (n= 10) while control mice (n = 10) were treated with IP injections of the corn oil vehicle. Mice were fed a western diet (TD88137, Harland Teklad, Madison, MI) throughout the protocol in attempts to accelerate atherosclerosis so that the effects of liver injury could be determined towards both atherosclerosis and thrombosis. In addition, both high fat diet and deficiency of apolipoprotein E have been shown to exacerbate liver injury induced by CCL4, possibly due to increased generation of oxysterols [9]. 24 hours following the last injection of drug or vehicle, alanine aminotransferase levels (9326.7 ± 2341.5 vs 39.0 ± 7.0 U/I, p b 0.002), lactate dehydrogenase levels (35110.7 ± 7538.0 vs 595.0 ± 57.8 U/I, p b 0.001) and monocyte chemoattractant protein-1 levels (504.5 ± 77.6 vs 165.0 ± 38.0 pg/ml, p b 0.005) were all elevated in CCL4 treated mice compared to vehicle treated mice, respectively. Histological analysis of the liver revealed greater macrophage content (4.0± 0.4 vs 0.7 ± 0.2% Mac3 positive staining area, p b 0.001, Fig. 1A and B), and increased collagen content (2.3 ± 0.4 vs 1.1 ± 0.3% positive staining area using Sirius red, p b 0.03) in mice treated with CCL4 compared to control mice. There were no differences in body weight throughout the protocol indicating CCL4 did not cause illness to the point of weight loss. Despite clear evidence of liver injury, there was no effect of chronic (6 weeks) CCL4 treatment on atherosclerosis surface area when measured by Oil-red-O (2.1 ± 0.3 vs 1.8 ± 0.3% surface coverage of aortic 0049-3848/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.thromres.2012.01.010
tree and major branches, p = NS) or by lesion cross sectional analysis at the aortic valve (1064.1 ± 159.5 vs 1142.2 ± 144.4 μm2, p = NS). To determine whether the liver injury induced by CCL4 would affect macrovascular arterial thrombosis at a distant site, photochemical injury was performed to the mid common carotid artery using Rose Bengal [11]. This model produces endothelial injury followed by occlusive thrombosis and the time to occlusion is used as a measure of thrombotic tendency [11]. Mice receiving CCL4 treatment formed occlusive thrombosis in a shorter period of time compared to vehicle control mice (Fig. 1C) indicating that chronic CCL4-induced liver injury leads to a prothrombotic state. This prothrombotic effect was independent of atherosclerosis as no differences in atherosclerosis were detected between mice receiving CCL4 or vehicle control. The relationship between liver disease and thrombosis is complicated as the liver is a source of inflammatory, coagulant, and anticoagulant factors [12]. Several types of liver diseases have been associated with both hemostatic defects and thrombosis [12]. In the case of steatohepatitis associated with obesity, the balance between bleeding and thrombosis is more in favor of thrombosis[13,14] and this may explain the increased risk of thrombotic events in patients with the metabolic syndrome. This study demonstrates that liver injury is sufficient to enhance thrombosis following arterial injury. Multiple factors may contribute to this risk including alterations in circulating procoagulant and antifibrinolytic factors. Interestingly, plasma levels of fibrinogen (Fg) (5.0 ± 2.0 vs 5.4 ± 1.1 mg/ml, p = NS) and plasminogen activator inhibitor-1 (PAI-1) (20.7 ± 10.3 vs 14.5 ± 3.4 ng/ml, p = NS) were not different between control mice and mice receiving CCL4, respectively. However, thrombinantithrombin complexes (TATs) were elevated in CCL4 treated mice compared to control mice (Fig. 1D) indicating that thrombin generation was increased (or anti-coagulant factors were decreased) in this model of steatohepatitis. Consistent with the hypothesis that thrombin generation is involved in steatosis, it was recently reported in a model of dietinduced obesity that TAT's are elevated with onset of steatosis [15]. Although this model of liver injury is not induced by obesity, it is an important step in determining the potential independent role of liver abnormalities on thrombosis, as models of obesity are confounded by many extrahepatic factors that could also contribute to thrombotic risk. However, even with this model we cannot rule out the possibility that extrahepatic effects of CCL4 are contributing to thrombosis. In this regard, it will be useful to determine the effects of other forms of hepatic injury on arterial thrombosis. In summary, hepatic steatosis induced by chronic CCL4 leads to enhanced arterial thrombosis. Changes in liver function coincident with disease states such as visceral obesity may contribute to the increased vascular risk associated with these diseases. Therapeutic strategies targeting coagulation-related pathways may be beneficial in reducing the prothrombotic state associated with steatohepatitis. Disclosure of conflict of interests The authors have no conflict of interests.
Letter to the Editors-in-Chief
A
Control injection
e167
B
CCl4 injection
Mac-3 staining (400x)
D *
80
*
3
TAT (ng/ml)
Time to Occlusion (min)
C
60 40 20 0
2 1 0
Control
CCl4
Control
CCl4
Fig. 1. Macropahge staining of liver tissue with Mac-3 anti-mouse antibody following (A) control oil or (B) CCL4 injection, magnification = 400x. Scale bar = 100 μm. (C) Bars represent time to occlusive carotid thrombosis in control mice (white bar) compare to mice receiving CCL4 (black bar) (mean ± SEM). *p b 0.003. (D) Bars represent thrombinantithrombin complexes in plasma from control (white bar) and CCL4-treated (black bar) mice (mean ± SEM). *p b 0.0001.
Acknowledgements This work was supported by the National Institutes of Health (HL073150 to D.T.E.) and the Veterans Administration Merit Review Award (1I01BX000353 to D.T.E.). References [1] Yusuf S, Hawken S, Ounpuu S, Bautista L, Franzosi MG, Commerford P, et al. Obesity and the risk of myocardial infarction in 27,000 participants from 52 countries: a case–control study. Lancet 2005;366:1640–9. [2] Yatsuya H, Folsom AR, Yamagishi K, North KE, Brancati FL, Stevens J. Race- and sex-specific associations of obesity measures with ischemic stroke incidence in the Atherosclerosis Risk in Communities (ARIC) study. Stroke 2010;41:417–25. [3] Borch KH, Braekkan SK, Mathiesen EB, Njolstad I, Wilsgaard T, Stormer J, et al. Anthropometric measures of obesity and risk of venous thromboembolism: the Tromso study. Arterioscler Thromb Vasc Biol 2010;30:121–7. [4] Zirlik A, Abdullah SM, Gerdes N, MacFarlane L, Schonbeck U, Khera A, et al. Interleukin-18, the metabolic syndrome, and subclinical atherosclerosis: results from the Dallas Heart Study. Arterioscler Thromb Vasc Biol 2007;27:2043–9. [5] Ridker PM, Buring JE, Cook NR, Rifai N. C-reactive protein, the metabolic syndrome, and risk of incident cardiovascular events: an 8-year follow-up of 14 719 initially healthy American women. Circulation 2003;107:391–7. [6] Ong JP, Elariny H, Collantes R, Younoszai A, Chandhoke V, Reines HD, et al. Predictors of nonalcoholic steatohepatitis and advanced fibrosis in morbidly obese patients. Obes Surg 2005;15:310–5. [7] Sun H, Koike T, Ichikawa T, Hatakeyama K, Shiomi M, Zhang B, et al. C-reactive protein in atherosclerotic lesions: its origin and pathophysiological significance. Am J Pathol 2005;167:1139–48. [8] Targher G, Bertolini L, Padovani R, Zenari L, Zoppini G, Falezza G. Relation of nonalcoholic hepatic steatosis to early carotid atherosclerosis in healthy men: role of visceral fat accumulation. Diabetes Care 2004;27:2498–500. [9] Ferre N, Martinez-Clemente M, Lopez-Parra M, Gonzalez-Periz A, Horrillo R, Planaguma A, et al. Increased susceptibility to exacerbated liver injury in hypercholesterolemic ApoE-deficient mice: potential involvement of oxysterols. Am J Physiol Gastrointest Liver Physiol 2009;296:G553–62.
[10] Luster MI, Simeonova PP, Gallucci RM, Matheson JM, Yucesoy B. Immunotoxicology: role of inflammation in chemical-induced hepatotoxicity. Int J Immunopharmacol 2000;22:1143–7. [11] Westrick RJ, Winn ME, Eitzman DT. Murine models of vascular thrombosis (Eitzman series). Arterioscler Thromb Vasc Biol 2007;27:2079–93. [12] Lisman T, Caldwell SH, Burroughs AK, Northup PG, Senzolo M, Stravitz RT, et al. Hemostasis and thrombosis in patients with liver disease: the ups and downs. J Hepatol 2010;53:362–71. [13] Cigolini M, Targher G, Agostino G, Tonoli M, Muggeo M, De Sandre G. Liver steatosis and its relation to plasma haemostatic factors in apparently healthy men–role of the metabolic syndrome. Thromb Haemost 1996;76:69–73. [14] Villanova N, Moscatiello S, Ramilli S, Bugianesi E, Magalotti D, Vanni E, et al. Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease. Hepatology 2005;42:473–80. [15] Kassel KM, Owens III AP, Rockwell CE, Sullivan BP, Wang R, Tawfik O, et al. Proteaseactivated receptor 1 and hematopoietic cell tissue factor are required for hepatic steatosis in mice fed a Western diet. Am J Pathol 2011;179:2278–89.
W. Luo M. Öhman A. Wright S. Kamrudin H. Wang C. Guo D. Eitzman⁎ University of Michigan, Department of Internal Medicine, Cardiovascular Research Center, Ann Arbor, Michigan, USA ⁎Corresponding author at: University of Michigan, Cardiovascular Research Center, 7301A MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109–0644, USA. Tel.: + 1 734 647 9865; fax: +1 734 936 2641. E-mail address: [email protected].(D. Eitzman) 28 November 2011